A Dose Ranging Pharmacokinetic Evaluation of IQP-0528 Released from Intravaginal Rings in Non-Human Primates.

PURPOSE: Design of intravaginal rings (IVRs) for delivery of antiretrovirals is often guided by in vitro release under sink conditions, based on the assumption that in vivo release will follow a similar release profile. METHODS: We conducted a dose-ranging study in the female reproductive tract of pigtail macaques using matrix IVRs containing IQP-0528, a poorly soluble but highly potent antiretroviral drug with an IC90 of 146 ng/mL. These IVRs consisted of drug-loaded segments, 15.6% IQP-0528 in Tecoflex 85A, comprising either all, half, or a quarter of the entire ring. RESULTS: In vitro release under sink conditions demonstrates loading-proportional release, with a cumulative 30-day release of 48.5 ± 2.2 mg for our 100% loaded ring, 24.8 ± .36 mg from our 50% loaded ring, and 13.99 ± 1.58 mg from our 25% loaded ring. In vivo, while drug concentration in vaginal fluid is well in excess of IQP-0528's EC90, we find no statistical difference between the different ring loadings in either swab drug levels or drug released from our rings. CONCLUSIONS: We show that in vitro release may not accurately reflect in vivo release, particularly for poorly soluble drugs. All tested loadings of our IVRs are capable of delivering IQP-0528 well in excess of the IC90.

Safety and Pharmacokinetics of Quick-Dissolving Polymeric Vaginal Films Delivering the Antiretroviral IQP-0528 for Preexposure Prophylaxis.

For human immunodeficiency virus (HIV) prevention, microbicides or drugs delivered as quick-dissolving films may be more acceptable to women than gels because of their compact size, minimal waste, lack of an applicator, and easier storage and transport. This has the potential to improve adherence to promising products for preexposure prophylaxis. Vaginal films containing IQP-0528, a nonnucleoside reverse transcriptase inhibitor, were evaluated for their pharmacokinetics in pigtailed macaques. Polymeric films (22 by 44 by 0.1 mm; providing 75% of a human dose) containing IQP-0528 (1.5%, wt/wt) with and without poly(lactic-co-glycolic acid) (PLGA) nanoparticle encapsulation were inserted vaginally into pigtailed macaques in a crossover study design (n = 6). With unencapsulated drug, the median (range) vaginal fluid concentrations of IQP-0528 were 160.97 (2.73 to 2,104), 181.79 (1.86 to 15,800), and 484.50 (8.26 to 4,045) µg/ml at 1, 4, and 24 h after film application, respectively. Median vaginal tissue IQP-0528 concentrations at 24 h were 3.10 (0.03 to 222.58) µg/g. The values were similar at locations proximal, medial, and distal to the cervix. The IQP-0528 nanoparticle-formulated films delivered IQP-0528 in vaginal tissue and secretions at levels similar to those obtained with the unencapsulated formulation. A single application of either formulation did not disturb the vaginal microflora or the pH (7.24 ± 0.84 [mean ± standard deviation]). The high mucosal IQP-0528 levels delivered by both vaginal film formulations were between 1 and 5 log higher than the in vitro 90% inhibitory concentration (IC90) of 0.146 µg/ml. The excellent coverage and high mucosal levels of IQP-0528, well above the IC90, suggest that the films may be protective and warrant further evaluation in a vaginal repeated low dose simian-human immunodeficiency virus (SHIV) transmission study in macaques and clinically in women.

Partial protection against multiple RT-SHIV162P3 vaginal challenge of rhesus macaques by a silicone elastomer vaginal ring releasing the NNRTI MC1220.

OBJECTIVES: The non-nucleoside reverse transcriptase inhibitor MC1220 has potent in vitro activity against HIV type 1 (HIV-1). A liposome gel formulation of MC1220 has previously been reported to partially protect rhesus macaques against vaginal challenge with a simian HIV (SHIV). Here, we describe the pre-clinical development of an MC1220-releasing silicone elastomer vaginal ring (SEVR), including pharmacokinetic (PK) and efficacy studies in macaques. METHODS: In vitro release studies were conducted on SEVRs loaded with 400 mg of MC1220, using simulated vaginal fluid (SVF, n = 4) and 1 : 1 isopropanol/water (IPA/H(2)O, n = 4) as release media. For PK evaluation, SEVRs were inserted into adult female macaques (n = 6) for 30 days. Following a 1 week washout period, fresh rings were placed in the same animals, which were then challenged vaginally with RT-SHIV162P3 once weekly for 4 weeks. RESULTS: SEVRs released 1.66 and 101 mg of MC1220 into SVF and IPA/H(2)O, respectively, over 30 days, the differential reflecting the low aqueous solubility of the drug. In macaque PK studies, MC1220 was consistently detected in vaginal fluid (peak 845 ng/mL) and plasma (peak 0.91 ng/mL). Kaplan-Meier analysis over 9 weeks showed significantly lower infection rates for animals given MC1220-containing SEVRs than placebo rings (hazard ratio 0.20, P = 0.0037). CONCLUSIONS: An MC1220-releasing SEVR partially protected macaques from vaginal challenge. Such ring devices are a practical method for providing sustained, coitally independent protection against vaginal exposure to HIV-1.

Safe and sustained vaginal delivery of pyrimidinedione HIV-1 inhibitors from polyurethane intravaginal rings.

The potent antiretroviral pyrimidinediones IQP-0528 (PYD1) and IQP-0532 (PYD2) were formulated in polyurethane intravaginal rings (IVRs) as prophylactic drug delivery systems to prevent the sexual transmission of HIV-1. To aid in the selection of a pyrimidinedione candidate and the optimal loading of the drug in the IVR delivery system, four pyrimidinedione IVR formulations (PYD1 at 0.5 wt% [PYD1(0.5 wt%)], PYD1(1 wt%), PYD2(4 wt%), and PYD2(14 wt%)) were evaluated in pigtail macaques over 28 days for safety and pyrimidinedione vaginal biodistribution. Kinetic analysis of vaginal proinflammatory cytokines, native microflora, and drug levels suggested that all formulations were safe, but only the high-loaded PYD2(14 wt%) IVR demonstrated consistently high pyrimidinedione vaginal fluid and tissue levels over the 28-day study. This formulation delivered drug in excess of 10 µg/ml to vaginal fluid and 1 µg/g to vaginal tissue, a level over 1,000 times the in vitro 50% effective concentration. The in vitro release of PYD1 and PYD2 under nonsink conditions correlated well with in vivo release, both in amount and in kinetic profile, and therefore may serve as a more biologically relevant means of evaluating release in vitro than typically employed sink conditions. Lastly, the pyrimidinediones in the IVR formulation were chemically stable after 90 days of storage at elevated temperature, and the potent nanomolar-level antiviral activity of both molecules was retained after in vitro release. Altogether, these results point to the successful IVR formulation and vaginal biodistribution of the pyrimidinediones and demonstrate the usefulness of the pigtail macaque model in evaluating and screening antiretroviral IVR formulations prior to preclinical and clinical evaluation.

Prodrug strategy for PSMA-targeted delivery of TGX-221 to prostate cancer cells.

TGX-221 is a potent, selective, and cell membrane permeable inhibitor of the PI3K p110ß catalytic subunit. Recent studies showed that TGX-221 has antiproliferative activity against PTEN-deficient tumor cell lines including prostate cancers. The objective of this study was to develop an encapsulation system for parenterally delivering TGX-221 to the target tissue through a prostate-specific membrane aptamer (PSMAa10) with little or no side effects. In this study, PEG-PCL micelles were formulated to encapsulate the drug, and a prodrug strategy was pursued to improve the stability of the carrier system. Fluorescence imaging studies demonstrated that the cellular uptake of both drug and nanoparticles was significantly improved by targeted micelles in a PSMA positive cell line. The area under the plasma concentration time curve of the micelle formulation in nude mice was 2.27-fold greater than that of the naked drug, and the drug clearance rate was 6.16-fold slower. These findings suggest a novel formulation approach for improving site-specific drug delivery of a molecular-targeted prostate cancer treatment.

Development of a Solid Supersaturable Micelle of Revaprazan for Improved Dissolution and Oral Bioavailability Using Box-Behnken Design.

PURPOSE: To enhance the oral bioavailability of revaprazan (RVP), a novel solid, supersaturable micelle (SSuM) was developed. METHODS: Surfactants and solid carriers were screened based on a solubility and a flowability test, respectively. Supersaturating agents, including Poloxamer 407 (P407), were screened. The SSuM was optimized using a Box-Behnken design with three independent variables, including Gelucire 44/14:Brij L4 (G44/BL4; X1) and the amounts of Florite PS-10 (FLO; X2) and Vivapur 105 (VP105; X3), and three response variables, ie, dissolution efficiency at 30 min (Y1), dissolution enhancing capacity (Y2), and Carr's index (Y3). The solid state property was evaluated, and a dissolution test was conducted. RVP, Revanex®, solid micelle (P407-free from the composition of SSuM), and SSuM were orally administrated to rats (RVP 20 mg equivalent/kg) for in vivo pharmacokinetic study. RESULTS: G44 and BL4 showed great solubility, with a critical micelle concentration range of 119.2-333.0 µg/mL. P407 had an excellent supersaturating effect. FLO and VP105 were selected as solid carriers, with a critical solidifying ratio (g/mL) of 0.30 and 0.91, respectively. With optimized values of X1 (-0.41), X2 (0.31), and X3 (-0.78), RVP (200 mg)-containing SSuM consisting of G44 (253.8 mg), BL4 (106.2 mg), FLO (99.3 mg), VP105 (199.8 mg), and P407 (40 mg) was developed, resulting in Y1 (40.3%), Y2 (0.008), and Y3 (12.3%). RVP existed in an amorphous state in the optimized SSuM, and the SSuM formed a nanosized dispersion in the aqueous phase, with approximately 71.7% dissolution at 2 h. The optimized SSuM improved the relative bioavailability of RVP in rats by approximately 478%, 276%, and 161% compared to raw RVP, Revanex®, and solid micelle, respectively. CONCLUSION: The optimized SSuM has great potential for the development of solidified formulations of poorly water-soluble drugs with improved oral absorption.

Antiretroviral release from poly(DL-lactide-co-glycolide) nanoparticles in mice.

OBJECTIVES: Free ritonavir, lopinavir and efavirenz injected intraperitoneally were compared with antiretroviral (AR) nanoparticles (NPs). METHODS: This is a prospective study in BALB/c mice comparing the pharmacokinetics of free drugs with AR NPs. All animals received free drugs or AR NPs (20 mg/kg) in PBS. In vitro replication assays were used for determination of the anti-HIV efficacy of NP formulations. At specific times (free drugs 0.08, 0.125, 0.25, 0.33, 1, 2 and 3 days; AR NPs 0.125, 0.33, 1, 2, 4, 7, 14, 21, 28, 35 and 42 days) mice were euthanized and serum and organs were harvested for determination of AR concentrations by HPLC. Single treatment of monocyte-derived macrophages (MDMs) infected with HIV-1(ada) compared AR NPs (0.005-0.05 mg/mL) with free efavirenz or lopinavir/ritonavir (0.01-0.1 mg/mL), blank NPs and controls. Results are presented as means ± SEM. RESULTS: Serum free AR drug concentrations peaked 4 h post-injection (ritonavir 3.9 ± 3.05, lopinavir 3.4 ± 2.5 and efavirenz 1.8 ± 0.63 µg/mL) and were eliminated by 72 h. Poly(dl-lactide-co-glycolide) NP animals had detectable ritonavir, lopinavir and efavirenz concentrations in all tissues for 28 days. Treatment of MDMs with AR NPs resulted in sustained inhibition of HIV-1(ada) replication. CONCLUSIONS: AR drug concentrations from NPs are sustained for 28 days in vivo and anti-HIV inhibition was comparable to that of free drugs in vitro and could be a sustained treatment for delivery of AR drugs.

Determination of mirodenafil and sildenafil in the plasma and corpus cavernous of SD male rats.

The purpose of the present study was to determine sildenafil and a novel PDE-5 inhibitor, mirodenafil in the plasma and corpus cavernosum tissue of rats to compare their pharmacokinetic properties. The concentrations of mirodenafil and sildenafil in the rat plasma and corpus cavernosum tissue samples were analyzed using LC-MS/MS after a single oral administration at a dose of 40mg/kg to rats. Although the T(max), Tlambda(1/2) and MRT were not different between mirodenafil and sildenafil, the C(max) and AUC of mirodenafil were significantly higher than those of sildenafil in the plasma and corpus cavernosum tissue. Consequently mirodenafil remained longer than sildenafil in the plasma and tissue. This may provide pharmacokinetic evidence for assessment of the in vivo efficacy of mirodenafil and sildenafil.

Comparison of a revaprazan-loaded solid dispersion, solid SNEDDS and inclusion compound: Physicochemical characterisation and pharmacokinetics.

The aim of this research was to compare three strategies for enhancing the solubility of poorly water-soluble revaprazan hydrochloride: solid dispersion, solid SNEDDS and inclusion compound. The influence of polymers, surfactants and oils on the drug solubility was assessed, and via the chosen carriers, the three types of formulations were prepared utilising spray drying technique. Their physicochemical properties, solubility, dissolution and pharmacokinetics in rats were performed compared with revaprazan powder. Among the liquid SNEDDS formulations assessed, the compositions of revaprazan, peceol, Tween 80 and Labrasol (10:15:55:30, weight ratio) provided the smallest emulsion size. Moreover, this liquid SNEDDS and dextran were suspended/dissolved in distilled water, and spray-dried, producing an optimal revaprazan-loaded solid SNEDDS. The appropriate solid dispersion and inclusion compound were composed of revaprazan, hydroxypropylmethylcellulose and cremophor A25 (5:1.4:5.6) and drug and hydroxyl-ß-cyclodextrin (2.5:8.77), respectively. The crystalline drug was converted to an amorphous state in all formulations. In the solid dispersion, the drug was attached to the hydrophilic carrier. The solid SNEDDS and inclusion compound contained aggregate microspheres and separate microspheres, respectively. All formulations significantly increased the drug solubility, dissolution, plasma concentration and AUC compared with revaprazan powder. These properties were ranked in the order solid dispersion ≥ solid SNEDDS > inclusion compound. Particularly, the solid dispersion improved about 9500-fold drug solubility and 10-fold oral bioavailability. Thus, the improved properties were considerably dependent upon these techniques, although all of the techniques employed similar mechanisms. Among the strategies checked, the solid dispersion system would be recommended as an oral revaprazan-loaded pharmaceutical product.

MRI Visualization of Injectable Ureidopyrimidinone Hydrogelators by Supramolecular Contrast Agent Labeling.

Information about the in vivo location, shape, degradation, or erosion rate of injected in situ gelating hydrogels can be obtained with magnetic resonance imaging (MRI). Herein, an injectable supramolecular ureidopyrimidinone-based hydrogel (UPy-PEG) is functionalized with a modified Gadolinium(III)-DOTA complex (UPy-Gd) for contrast enhanced MRI. The contrast agent is designed to supramolecularly interact with the hydrogel network to enable high-quality imaging of this hydrogel. The applicability of the approach is demonstrated with successful visualization of the Gd-labeled UPy-PEG hydrogel after targeted intramyocardial catheter injection in a pig heart.

pH, serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures.

It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8+/-0.1 and 7.4+/-0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0-2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8+/-0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00-0.30 mol l(-1)). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01-0.30 mol l(-1). The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study.

Magnetic-luminescent YbPO4:Er,Dy microspheres designed for tumor theranostics with synergistic effect of photodynamic therapy and chemotherapy.

In this paper, magnetic and fluorescent bifunctional YbPO4:Er,Dy microspheres were synthesized via a simple solvothermal method. The prepared microspheres exposed to 980 nm near-infrared (NIR) laser light emitted bright upconversion fluorescence (450-570 nm) after calcination at high temperatures (>800°C). Results of magnetic resonance studies demonstrated that the YbPO4:Er,Dy microspheres are more suitable to be used as a transverse relaxation time (negative) contrast magnetic resonance imaging agent. The microspheres successfully entered the human hepatocellular carcinoma cells and presented low toxicity. A well-selected photodynamic therapy (PDT) drug, merocyanine 540 (MC540) with an ultraviolet-visible spectroscopy absorption maximum of 540 nm, was loaded onto the microspheres to obtain YbPO4:Er,Dy-MC540. Since the upconversion fluorescence emitting from the microspheres could be absorbed by MC540 with a small absorption/emission disparity, YbPO4:Er,Dy-MC540 could kill the hepatocellular carcinoma cells via PDT mechanism effectively. In other words, being upconverting particles, the prepared microspheres acted as light transducers in the NIR light-triggered PDT process. A chemotherapy drug, doxorubicin, was further loaded onto YbPO4:Er,Dy-MC540 to achieve enhanced antitumor effect based on synergistic therapeutic efficacy of PDT and chemotherapy. It is expected that the prepared YbPO4:Er,Dy microspheres have applications in tumor theranostics including magnetic-fluorescent bimodal imaging and NIR light-triggered PDT.

Determination of lopinavir and ritonavir in blood plasma, seminal plasma, saliva and plasma ultra-filtrate by liquid chromatography/tandem mass spectrometry detection.

A method based on liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the simultaneous determination of lopinavir (LPV) and ritonavir (RTV) in human blood, semen and saliva samples. The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions: m/z 629 > 447.1 for LPV, 721.18 > 268.02 for RTV and m/z 747.22 > 322.03 for the internal standard (IS). The limit of quantification was 1 ng/mL for both analytes in all matrices. The method was linear in the studied range (1-2000 ng/mL for LPV and 1-200 ng/mL for RTV), with r2 > 0.99 for each drug, and the run time was 4.5 min. The intra-assay precisions (%) were in the ranges of 0.1-14.2 (LPV) and 0.4-12.7 (RTV), the inter-assay precisions were in the ranges of 2.8-15.3 (LPV) and 1.1-12.8 (RTV) and the intra-and inter-assay recoveries were >85% for both drugs. The extraction efficiencies were 73.5-118.4% for LPV and 74.4-126.2% for RTV. The analytical method was applied to measure LPV and RTV concentrations in blood plasma (total and unbound fraction), saliva and semen of six HIV+ individuals under stable treatment with Kaletra soft gel capsules. The results were consistent with previously published data.

Merocyanine-sensitized photoinactivation of enveloped viruses.

A wide range of enveloped viruses, including human herpes simplex virus type 1, human cytomegalovirus, human T cell leukemia/lymphoma virus type I, human immunodeficiency virus type 1, Sindbis virus, and Friend erythroleukemia virus, are highly susceptible to merocyanine 540 (MC 540)-sensitized photoinactivation. By contrast, human pluripotent hematopoietic stem cells, red cells, factor VIII, and von Willebrand factor are much less sensitive. This suggests that MC 540 may be useful for the inactivation of enveloped viruses in blood and blood products. The dye has a low acute systemic toxicity, is rapidly eliminated from the blood stream, and has little or no mutagenic potential. The currently available data support the view that MC 540-sensitized photo-inactivation interferes with early events in the infectious process, notably the ability of the virus to adhere to and penetrate its host cell. The viral envelope is a major target of photodynamic damages which appear to be mediated at least in part by singlet molecular oxygen.